Nipping disease in the bud: nSMase2 inhibitors as therapeutics in extracellular vesicle-mediated diseases

Abstract

Extracellular vesicles (EVs) are indispensable mediators of intercellular communication, but they can also assume a nefarious role by ferrying pathological cargo that contributes to neurological, oncological, inflammatory, and infectious diseases. The canonical pathway for generating EVs involves the endosomal sorting complexes required for transport (ESCRT) machinery, but an alternative pathway is induced by the enrichment of lipid membrane ceramides generated by neutral sphingomyelinase 2 (nSMase2). Inhibition of nSMase2 has become an attractive therapeutic strategy for inhibiting EV biogenesis, and a growing number of small–molecule nSMase2 inhibitors have shown promising therapeutic activity in preclinical disease models. This review outlines the function of EVs, their potential role in disease, the discovery and efficacy of nSMase2 inhibitors, and the path to translate these findings into therapeutics.

Introduction: extracellular vesicles

EVs are small, membrane-enclosed vesicles ranging in size from 30 nm to several microns in diameter. Although the minimum size of an EV is defined by the physical constraints of membrane composition and curvature, the maximum size is limited by its biogenesis pathway [1]. Exosomes arise from the inward budding of the membrane of the multivesicular body (MVB) to form intraluminal vesicles (ILVs), which are released into the extracellular space as exosomes upon MVB–plasma membrane fusion [1] (Figure 1). Whereas exosomes are limited in size by the dimensions of the MVB, ectosomes, also called microvesicles or microparticles, bud outward from the plasma membrane. Ectosomes can be as small as exosomes, but can also reach multiple microns in diameter [2]. The isolation of specific EV subtypes is difficult, beause there are no reliable universal markers, and each has overlapping characteristics [3]. Each isolation technique has a bias toward different subsets of EVs and varying levels of non-EV contaminants, further complicating efforts to define EV populations (reviewed in [4]). Novel naming conventions instead focus on the general size or other physical characteristics of the EV, such as subdividing classes into small vesicles (<200 nm) and medium/large vesicles (>200 nm). In this review, we follow the guidelines established in [3] and use the general term ‘EV’.

Figure 1.

Schematic of extracellular vesicle (EV) biogenesis, cargo content, and physiological roles. EV biogenesis begins with the formation of interluminal vesicles (ILVs) to generate multivesicular bodies (MVBs) via either an endosomal sorting complexes required for transport (ESCRT)-dependent or -independent pathway. (a) ESCRT-dependent biogenesis requires the involvement of the ESCRT machinery to assist in the inward budding and pinching off of the ILVs. (b) ESCRT-independent biogenesis relies on increased ceramide concentrations generated by neutral Sphingomyelinase 2 (nSMase2), causing the membrane to bud inward to form ILVs. (c) MVBs contain many ILVs that can be targeted towards the plasma membrane for extracellular release. (d) The MVB fuses with the plasma membrane and releases the ILVs, which upon extracellular release become EVs. (e) EVs can carry a variety of cargo, both cytosolic and membrane associated, including lipids, proteins, and nucleic acids. (f) The biological consequences of EV release can be physiological, such as the elimination of cellular waste and cell-cell communication, or pathological, where disease-associated cargos contribute to cancer, inflammation, neurodegenerative disease, and infectious disease progression. Abbreviation: SM, sphingomyelin. Figure created with BioRender (BioRender.com).

The beginnings of modern EV research are often traced to important studies during the 1980s, when the groups of Trams, Johnstone, and Stahl discovered that cells use EVs to shed certain proteins [5-7]. However, hints of the diverse functionality of EVs in intercellular communication had been present in the literature for decades prior [8], ranging from involvement of platelet releasates in coagulation [9] to the membranous mating-type ‘gamones’ of Chlamydomonas [10] to bone EV-mediated calcification [11]. However, establishment of a strong field of study would wait until evidence emerged during the early 2000s that EVs could function similar to viruses in transferring genetic information between cells [12]. We now understand that EVs have broad and crucial roles in maintaining normal cell health and function through long- and short-range intercellular communication.

Extracellular vesicle biogenesis

EVs contain a variety of cargo, including proteins, nucleic acids, and bioactive lipids, which are packaged from the original cell and travel through the extracellular space, or broadly through bodily fluids, before being incorporated into recipient cells. In part because of the heterogeneity of the cargo, the mechanisms responsible for sorting specific cargo into EVs and how they are then targeted to appropriate distal destinations are not fully understood, and are an active area of research.

Diverse EV biogenesis mechanisms are found at different subcellular sites. There are two major pathways identified for EV biogenesis originating from the MVB. The first to be discovered was the ESCRT pathway, which comprises four distinct subcomplexes of ESCRT-0, -I, -II, and –III, together with the ATPase vacuolar protein sorting-associated protein 4 (VPS4). Each of these has distinct and sequential roles, including initiating ILV budding (-0), binding cargo (-I, and -II), vesicle maturation and constriction (-III), and membrane scission of the ILV (VPS4) [13]. Several subunits and accessory proteins are used as EV markers, such as ALG-2 interacting protein X (Alix), an ESCRT-III accessory protein that stabilizes the Snf7 subunit, and Tumor susceptibility gene 101 (Tsg101), an ESCRT-I subunit thought to bind ubiquitinated cargo[13]. In 2008, it was discovered that EVs can also be produced in the absence of ESCRT proteins, which led to the identification of a secondary, ceramide-dependent pathway [14]. A major source of ceramide is the hydrolysis of sphingomyelin (SM) into ceramide and phosphorylcholine by members of the sphingomyelinase (SMase) enzyme family (Figure 1a). Important biophysical properties of ceramide are its marked intrinsic negative curvature because of a small polar head group and its tendency to segregate into ceramide-enriched microdomains that facilitate the transition of the membrane from a more linear lamellar phase to a more densely packed and rounded hexagonal phase [15]. A focal enrichment of ceramide at the membrane facilitates the formation of ILVs [14]. More recently, evidence for a novel third pathway of ILV budding, independent from both the ESCRT and ceramide pathways, involving ADP ribosylation factor 6 (ARF6) and phospholipase D2 (PLD2), has also been reported [16]. EVs that originate from the plasma membrane come about via rearrangement of cytoskeletal components and recruitment of proteins, which aid in the outward budding and scission of the vesicle into the extracellular space [2].

The sphingomyelinase family

Sphingomyelinases are a diverse family of enzymes responsible for catalyzing the hydrolysis of SM to ceramide and phosphorylcholine. The primary classification of these enzymes is based on their optimal enzymatic pH, cation requirement, and localization, and include lysosomal acidic sphingomyelinase (L-aSMase), secreted zinc-dependent acidic sphingomyelinase (S-aSMase), alkaline sphingomyelinase (alkSMase), and magnesium-dependent and independent neutral sphingomyelinase (nSMase).

aSMase is encoded by the sphingomyelinase phosphodiesterase 1 (SMPD1) gene and was first described in 1963 by Shimon Gatt [17]. Functioning at an optimal pH of 5, aSMase is widely expressed in mammalian tissues and is either localized in the lysosome or secreted. There is an inherited mutant form of aSMase that causes toxic accumulation of SM, leading to a lysosomal storage disorder called Niemann–Pick disease.

alkSMase was first described in 1969 by Åke Nilsson [18]. The enzyme was initially isolated from human duodenal contents and pig intestinal homogenates, where it had the highest level of activity at an alkaline pH of 9. Despite its SMase activity, its amino acid sequence is dissimilar from that of other SMases and more closely matches the nucleotide pyrophosphatase/phosphodiesterase (NPP) family, hence its gene name is ENPP7 [19]. Its subcellular localization is mainly to the plasma membrane and Golgi apparatus, and its tissue expression is enriched in intestinal mucosa. AlkSMase has not been as extensively studied as the other SMases, but a decrease in its expression has been linked to gastrointestinal (GI) cancers and inflammatory bowel disease [19].

nSMase was first described in 1967 by Schneider and Kennedy as a Mg²⁺-dependent SMase optimally active at pH 7.4 isolated from spleens of patients with Niemann-Pick disease [20]. Researchers then identified additional Mg²⁺-dependent SMases from human and rat brain tissue [21]. To date, four cation-dependent isoforms of nSMases have been discovered: nSMase 1, 2, 3, and mitochondrial-associated nSMase (MA-nSMase), which are encoded by SMPD2–5, respectively [22,23]. Although a Mg²⁺-independent cytosolic form of nSMase was described, little is known about it other than it is activated by vitamin D3 in vitro [24]. Whereas nSMase2 has been extensively studied, less is known about nSMase 1, 3 and MA-nSMase. nSMase1 is expressed ubiquitously, with protein localization in the Golgi apparatus and endoplasmic reticulum (ER). Its physiological role remains unknown, because neither nSMase1 knockout (KO) nor overexpression significantly impacts lipid storage or metabolism [23]. nSMase3 does not share sequence homology with nSMase 1 or 2, and is highly expressed in skeletal and cardiac muscle [23]. Similar to nSMase1, its function is poorly elucidated. MA-nSMase was initially identified in zebrafish, but has since been observed in murine brain, testis, pancreas, and epididymis [22]. It is not currently known whether MA-nSMase is expressed in human tissues. In vitro overexpression results in increased SMase activity and ceramide production, indicating functional enzymatic activity, but little else is known at this time [22].

By contrast, nSMase2 has been widely studied. It is highly expressed in the plasma membrane and Golgi apparatus of many mammalian cell types, with highest expression in the brain. nSMase2 has been implicated in the cellular stress response, ceramide-mediated apoptosis, inflammation, and EV biogenesis [14,23]. Unlike nSMase1-KO mice, nSMase2-KO mice exhibit substantial reductions in SMase activity and have a dwarfism phenotype, with skeletal defects and growth hormone deficits [25]. The role of nSMase2 in bone structure is further supported by fro/fro mice, which have an autosomal recessive mutation in SMPD3, causing significant attenuation of nSMase activity and characteristics similar to those taht arise in autosomal recessive forms of osteogenesis imperfecta [26]. Based on evidence demonstrating a significant reduction in total SMase activity in both the KO and mutant nSMase2 animals, nSMase2 appears to the predominant source of nSMase in the body and, consequently, has the potential to serve as an effective therapeutic target in several disease areas where ceramide-mediated release of EVs has a key pathogenic role. Here, we provide an overview of the recent advances made in the field, in particular with respect to the therapeutic utility of nSMase2 inhibition.

Evolution of neutral sphingomyelinase 2 inhibitors

Background

Since the discovery of SMase activity in various mammalian tissues with neutral optimal pH, several structurally diverse molecules have been reported as inhibitors of this enzymatic activity. Most of the early work on nSMase inhibitors was conducted using mammalian tissues as sources of the enzyme. Given the lack of knowledge regarding the existence of nSMase isoforms at the time, these inhibitors were reported as nSMase inhibitors. However, in this review, these compounds are referred to as nSMase2 inhibitors given that the nSMase activity of these tissues is predominantly derived from this isoform. Several review articles have been written at different times to capture the progress made in the field of nSMase2 inhibitors [27-30]. Hence, this section focuses on the representative nSMase2 inhibitors (Table 1), including the latest generation of nSMase2 inhibitors with greater translational potential.

Table 1.

Representative nSMase2 inhibitors

Compound	IC50 (mM)	Enzyme source	Mode of inhibition	Origin of discovery	Refs
	1	Rat brain	Mixed	Microbial screening	[32]
	2.8	Rat brain	Unknown	Sphingomyelin analog	[33]
	1.7 (Ki value)	Bovine brain	Competitive	Ceramide analog	[34]
	1	Rat brain	Noncompetitive	High-throughput screening	[35]
	5	Human recombinant	Noncompetitive	High-throughput screening	[36]
	0.03	Human recombinant	Noncompetitive	High-throughput screening	[37]
	0.3	Human recombinant	Noncompetitive	High-throughput screening followed by medicinal chemistry efforts	[38]
	0.5	Human recombinant	Noncompetitive	High-throughput screening followed by medicinal chemistry efforts	[39]

Microbial products

Early efforts on the search of nSMase2 inhibitors concentrated on screening of extracts containing microbial products. Although most compounds identified through this approach were reported to be relatively weak inhibitors (IC₅₀ >10 μM), scyphostatin 1 (Table 1) isolated from Trichopeziza mollissima displayed potent nSMase2 inhibition with an IC₅₀ value of 1.0 μM using rat brain microsomes as an enzyme source [31,32]. In the subsequent biological evaluation, scyphostatin showed ~50-fold selectivity over aSMase and inhibited lipopolysaccharide (LPS)-induced production of prostaglandin E2 and IL-1β in human monocytes [31]. Despite the potent inhibitory activity and promising biological profile, the utility of scyphostatin was limited because of its poor aqueous solubility and chemical instability [32]. Efforts to develop synthetic derivatives of scyphostatin failed to identify nSMase2 inhibitors with comparable potency.

Sphingomyelin and ceramide derivatives

Another approach undertaken to identify nSMase2 inhibitors was to explore synthetic derivatives of sphingomyelin and ceramide, the substrate and product of nSMase2-catalyzed reactions. For instance, compound 2 (Table 1) is a sphingomyelin analog with a carbamate moiety as a replacement for the phosphodiester group. It inhibited nSMase2 from rat brain microsomes with an IC₅₀ value of 2.8 μM [33]. Despite its sphingomyelin-like structure, no significant inhibition of aSMase and Bacillus cereus SMase was achieved by compound 2 at concentrations up to 100 μM [33]. KY3535 (compound 3, Table 1) is a ceramide analog where the fatty acid amide group is replaced by a thiourea group [34]. It was found to inhibit partially purified bovine brain nSMase2 in a competitive manner with a Kⁱ value of 1.7 μM. However, its selectivity over aSMase was not reported.

Pharmacological effects of compound 2 and KY3535 were not investigated in vivo. One possible reason is the lack of desirable absorption, distribution, metabolism, and excretion (ADME) properties because of the presence of a long carbon chain contributing to the high lipophilicity of these compounds.

nSMase2 inhibitors identified from screening

Screening of compound libraries was another approach explored by many groups in an attempt to identify nonlipid-like nSMase2 inhibitors. GW4869 (compound 4, Table 1) was identified by a high-throughput assay using a partially purified rat brain nSMase as a source of enzyme and radiolabeled sphingomyelin as a substrate [35]. GW4869 was found to be a noncompetitive inhibitor, with an IC₅₀ value of 1 μM and no inhibitory activity against aSMase. Since it was first reported, GW4869 has served as a tool compound in several preclinical studies as described later. However, GW4869 was not further developed as a clinical candidate or used as a molecular template for further structural optimization because of its highly lipophilic core structure unsuitable to serve as a tractable lead.

The first nSMase2 screening assay with human recombinant enzyme led to the discovery of cambinol (compound 5, Table 1) as a potent nSMase2 inhibitor [36]. It exhibited a noncompetitive mode of inhibition with an IC₅₀ value of 7 μM. Cambinol was found to be equally potent (IC₅₀ = 6 μM) against the rat nSMase2, enabling more rigorous cross-species translational studies.

The screening method that identified cambinol was fluorescence based and more practical than radiosubstrate-based methods that had been predominantly used in nSMase2 assays previously. Indeed, the new screening assay method was further miniaturized into a 1536-well format, enabling a screening of >350 000 compounds in collaboration with National Center for Advancing Translational Sciences (NCATS). These efforts led to the discovery of 2,6-dimethoxy-4-(5-phenyl-4-thiophen-2-yl-1H-imidazol-2-yl)-phenol (DPTIP, compound 6, Table 1) with an IC₅₀ value of 30 nM [37], representing one of the most potent nSMase2 inhibitors identified to date. DPTIP was one of the first nSMase2 inhibitors evaluated for ADME properties. It showed metabolic stability in both mouse and human liver microsomes, but unfortunately exhibited poor oral bioavailability. However, following intraperitoneal injection (10 mg/kg) in mice, DPTIP achieved a brain-to-plasma ratio of 0.26, with brain levels above its IC₅₀ value for 4 h post injection.

nSMase2 inhibitors derived from screening hits

Additional screening efforts using the same assay method followed by extensive medicinal chemistry efforts led to the discovery of phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)-carbamate (PDDC, compound 7, Table 1) [38], a noncompetitive nSMase2 inhibitor with an IC₅₀ value of 300 nM. Although PDDC exhibited tenfold weaker potency over DPTIP, it showed excellent overall ADME properties. It was found to be stable in both mouse and human microsomes. Furthermore, it showed excellent oral bioavailability (F%=88) and brain penetration (a brain-to-plasma ratio of 0.6) in mice, providing the potential to serve as a valuable tool compound to investigate the therapeutic utility of nSMase2 inhibition in various preclinical models, including neurological disorders.

Another screening campaign of a ~70 compound library largely comprising fused indolines followed by structural optimization efforts, which led to the discovery of compound 8 (Table 1), a dual nSMase2 and acetylcholinesterase (AChE) inhibitor with IC₅₀ values of 500 nM and 1.7 mM, respectively [39]. Following subcutaneous injection (20 mg/kg) in mice, the average brain level of compound 8 at the peak was 262 ng/g (~0.8 μM), exceeding its IC₅₀ value for nSMase2.

The modular nature of compounds DPTIP, PDDC, and compound 8 can be exploited for further systematic structural optimization by fine-tuning various parts of the parent molecules. Indeed, preliminary efforts along this line have already been attempted for DPTIP and PDDC [40,41].

Conclusion

Judging from the latest progress, the tremendous efforts devoted to the discovery of nSMase2 inhibitors over the past decades have begun to bear fruit, with several structurally distinct inhibitors serving as tractable leads for further optimization. DPTIP, PDDC, and compound 8 are particularly promising because they have already shown utility as in vivo probe molecules, as described later. New nSMase2 inhibitors emerging from these leads should be well positioned to accelerate therapeutic development, given the growing evidence demonstrating the therapeutic utility of nSMase2 inhibition, as detailed in the following sections.

Utility of nSMase2 inhibition in neurodegenerative diseases

Neurodegenerative disease was one of the first areas where nSMase2 inhibition was explored as a therapeutic approach. Increased ceramide levels have been observed in patients with Alzheimer’s Disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) [42]. New insights into the mechanisms underlying these diseases indicate that EV-mediated transmission of toxic proteins might be associated with disease propagation; thus, nSMase2 inhibition of EV biogenesis was proposed as a therapeutic strategy. Several studies using pharmacological inhibition or genetic knockdown have been instrumental in demonstrating the potential of nSMase2 inhibition for the treatment of neurodegenerative diseases, detailed herein (Table 2).

Table 2.

Utility of nSMase2 inhibition/KO in preclinical models of neurological disorders

Disease	Model	Result of nSMase2 inhibition/KO	Refs
AD	5XFAD mice	Decreased serum EV levels, brain ceramide levels, glial activation, tau phosphorylation, and Aβ plaque load, improved memory	[43]
	fro;5XFAD mice	Reduction in EV release in brain and blood, decreased ceramide levels and Aβ plaque load	[44]
	5XFAD mice	Improved body weight and memory	[41]
	AAV-tau or PS19 mice	Reduced tau spread from entorhinal cortex to hippocampus in AAV-tau mice; reduced hippocampal tau in PS19 mice	[45]
	Synapatosomes from human AD brain	Reduced EV release and tau spread	[46]
	HEK293T tau RD P301S cells; P301S + ICV IL-1β in mice	Reduced tau spread in vitro; decreased brain-derived EV levels and lowered tau levels in brain-derived EVs in vivo	[39]
ALS/FTD	Primary rat motor neuron cells	Prevented NGF-induced motor neuron death	[54]
MS	Cuprizone-fed mice	Inhibited elevated posterior corpus callosum nSMase2 activity and ceramide levels, improved myelination status	[61]
DMD	mdx:nSMase2 DKO mice; H2K cells	Reduced anxiety behavior and muscle inflammation, enhanced muscle function in DKO mice; enhanced survival in H2K cells with inhibitor	[60]
PD	SH-SY5Y neuroblastoma cells	Decreased secretion of γ-synuclein	[49]
		Decreased transfer of α-synuclein between cells	[50]
	Midbrain slice cultures	Decreased EV release and IFN-γ/LPS-induced dopaminergic neurodegeneration	[51]
Prion disease	GT1-7 hypothalamic cells	Reduced EV biogenesis and PrP packaging	[57,62]
	Mov neuroglial cells	Decreased release of PrP	[58]

Alzheimer’s disease

Recent evidence implicated EV-dependent propagation of amyloid β (Aβ) and hyperphosphorylated tau within the AD brain. These data led to a proposed role for nSMase2 inhibition in ameliorating AD pathogenesis. The first in vivo assessment of nSMase2 inhibition in AD mice occurred in the Bieberich laboratory [43], where systemic GW4869 administration in 5XFAD mice markedly reduced brain ceramide levels, EV release, brain Aβ_1-42, and amyloid plaques. This pharmacological inhibition replicated the findings of their earlier genetic study, in which nSMase2-deficient fro/fro mice crossed with 5XFAD AD mice (fro;5XFAD) had reduced amyloid plaques, decreased EVs, and an improvement in fear-conditioning behavior compared with 5XFAD wild-type mice [44]. Recently, these findings were reproduced using the nSMase inhibitor PDDC, demonstrating drug-induced improvements in body weight and performance in contextual and cued fear-conditioning assays in 5XFAD mice [41].

The effect of nSMase2 inhibition on EV-mediated tau propagation has also been investigated. The Ikezu lab was the first to demonstrate the utility of inhibiting EV release to slow tau propagation in an adeno-associated virus (AAV) mouse model of rapid tau propagation. They reported that tau spreads along connectivity pathways in the brain via an EV-dependent mechanism, which was attenuated by either genetic knockdown or pharmacological inhibition of nSMase2 [45]. Further studies utilizing synaptosomes isolated from human AD brain and naïve recipient cells demonstrated that the nSMase2 inhibitors cambinol and GW4869 reduced EV production and limited the spread of tau between donor and recipient cells [46]. The same team developed a carbamate furoindoline compound (compound 8 in Table 1) with dual nSMase2/AChE inhibitory activity, which significantly reduced the levels of tau cargo in brain-derived EVs isolated from P301S AD mice challenged with an intracerebroventricular (ICV) injection of IL-1β [39]. These data are translationally relevant because the presence of phosphorylated tau, as well as Aβ, in neuronally derived EVs isolated from plasma from patients with AD were found to be a strong predictor of AD disease progression [47].

Parkinson’s disease

Mounting evidence demonstrates that α-synuclein, a main component of the abnormal Lewy body aggregates in PD, propagates interneuronally in the brain via EVs [48]. GW4869 was shown to inhibit the secretion of γ-synuclein, an upstream stimulator of α-synuclein seeding [49]. More recently, cambinol and nSMase2 knockdown were demonstrated to inhibit EV release and block the transfer of human α-synuclein from donor SH-SY5Y neuronal cells to co-cultured naïve recipient cells [50]. These findings were supported by a separate study showing that GW4869-mediated inhibition of EV release from rat brain slice cultures resulted in reduced cell death following interferon (IFN)-γ/lipopolysaccharide (LPS) treatment [51]. Additionally, clinical data demonstrated that higher α-synuclein levels in plasma-derived neuronal EVs in patients with PD were associated with higher risk for motor symptom worsening. These data also raise the possibility that EV contents could serve as an accessible biomarker for PD disease progression [52].

Amyotrophic lateral sclerosis and frontotemporal dementia

EV-mediated transport of pathological proteins has also been implicated in ALS and frontotemporal dementia (FTD). Both transactive response (TAR) DNA-binding protein 43 (TDP-43) and superoxide dismutase 1 (SOD1), the main aggregate-forming proteins in patients with ALS/FTD, have been identified as EV cargo, where they have demonstrated the ability to seed misfolding in naïve recipient cells [53]. Primary rat motor neurons overexpressing mutant SOD1^G93A exhibited reduced cell death in response to nerve growth factor (NGF) and nitric oxide (NO) when simultaneously treated with nSMase2 inhibitors [54]. In models of TDP-43 inclusions, the results were less clear. Whereas TDP-43 was present in EVs released from hTDP-43-containing Neuro2a cells as well as in brains from patients with ALS [55], GW4869 treatment in Neuro2a cells caused TDP-43 inclusion increases, not decreases. In addition, GW4869-treated TDP-43^A315T mice exhibited worse motor deficits, increased muscle denervation, and enhanced cognitive deficits [55]. These studies highlight the need for further experimental clarification of the role of nSMase2 in ALS/FTD.

Prion disease

Prion diseases have also garnered increased interest in the therapeutic potential of nSMase2 inhibitors. In fact, the ‘prion-like’ theory of neurodegenerative diseases arose from the discovery that seed-capable prion proteins are packaged into EVs that are able to induce misfolding in recipient cells [53,56]. Despite the early understanding of the importance of EVs, there have only been a few studies conducted with nSMase2 inhibitors in prion disease models. The Hill laboratory showed that GW4869 treatment and small interfering (si)RNA knockdown of nSMase2 in GT1–7 neuronal cells infected with mouse-adapted human prion protein (PrP) reduced the amount of both total PrP and disease-associated PrP [57]. A separate study confirmed that nSMase inhibition was capable of reducing the amount of EV-associated PrP [58]. Although additional work is needed, these initial studies demonstrate a possible role for nSMase2 inhibition in prion diseases.

Other degenerative diseases

nSMase2 inhibition has also been implicated in other degenerative diseases, such as multiple sclerosis (MS) and Duchenne muscular dystrophy (DMD). Elevated ceramide levels have been observed in patients with MS, with a recent report highlighting the pathogenic role of nSMase2 in oligodendrocytes by showing that cambinol promotes remyelination in a mouse model of MS [59]. In DMD, knocking out nSMase2 in a mouse model reduced inflammation, enhanced muscle function, and alleviated some anxiety-like behaviors [60]. Interestingly, the authors also observed increased EVs in the media of H2K DMD myoblasts compared with normal mouse myoblasts and prolonged survival of H2K myoblasts with increasing concentrations of GW4869, suggesting that EV release is detrimental.

Conclusion

Although much of the work investigating nSMase2 inhibitors in neurological disorders has focused on AD, a growing number of neurological disorders are now being linked to abnormal nSMase2 function. These promising data highlight the importance of identifying potent, brain-penetrable nSMase2 inhibitors that could be translated into clinical studies.

Utility of nSMase2 inhibition in cancer

Given the role of ceramide in inducing apoptosis and growth arrest, as well as the location of nSMase2 on chromosome 16q22.1, a region commonly lost in prostate and breast cancer, nSMase2 was originally thought to have a tumor-suppressor role (reviewed in [63]). Recently, however, a growing body of evidence has implicated EVs in propagating tumorigenesis, metastasis, drug resistance, and immunotherapy [63]. This has led to renewed interest in nSMase2 inhibitors as potential cancer therapeutics (Table 3).

Table 3.

Utility of nSMase2 inhibition/KO in preclinical models of cancer

Cancer	Model	Result of nSMase2 inhibition	Refs
Breast	Cal51 cells	Decreased tumor cell senescence induced by paclitaxel	[76]
	4T1 cells in mice	Inhibited tumor growth and augmented anti-PD-L1 therapeutic effect	[84]
Cervical	HeLa cells	Enhanced antimigration effects of mifepristone, decreased cell viability, and enhanced apoptosis	[70]
	HT29 and FHC cells	Attenuated CRC-derived EV-mediated migration of normal colon epithelial cells	[74]
CRC	LoVo cells in mice	Suppressed tumor growth	[68]
	HT29 and HCT116 cells	Inhibited hypoxic EV-mediated migration and invasion	[69]
	SW480 cells	Decreased oxaliplatin resistance	[79]
Duodenal	AZ-Pa7 cells	Decreased accumulation of toxic polyadenylate-binding protein 1 in EVs	[65]
Gastric	M2 macrophages and MFC cells; MFC cells in mice	Inhibited M2 macrophage-mediated migration; decreased MFC metastasis	[72]
	SCG7901/ADR cells	Decreased doxorubicin resistance	[80]
Lung	HMEC-1 and A549 cells	Inhibited EV containing tumor conditioned media-mediated transformation of epithelial cells to cancer-like phenotype	[64]
	A549 cells and cancer associated fibroblasts	Inhibited EV release by CAFs, prevented epithelial to mesenchymal transition	[71]
	HBMECs and NCI-H446 cells	Decreased metastasis markers in SCLC cells	[73]
Multiple myeloma	5TGM1 cells in mice	Blocked bortezomib resistance	[78]
Oral	HSC-3-R and SCC-9-R cells	Ameliorated cisplatin resistance	[77]
Pancreatic	PANC-1 cells	Inhibited proliferation, migration, and chemokine expression	[66]
	CAF and AsPC1 cells; CAF and AsPC1 cells in NOD-SCID mice	Decreased survival of PDAC cells; slowed tumor growth	[75]
Prostate	C4-2B-R, CWR-R1-R, and LNCaP-R cells	Attenuated enzalutamide resistance	[81]
	TRAMPC2 cells in mice	Inhibited tumor growth via nSMase2-null tumors	[85]
Skin	B16BL6 cells in vitro and in mice	Inhibited EV release and decreased proliferation; suppressed tumor growth	[67]

Tumorigenesis and metastasis

Tumor-derived EVs facilitate tumor metastasis and transfer their content from parent tumor cells to naïve cells, leading to malignant transformations. EVs from tumor-conditioned media in vitro can transform normal endothelial cells into a proangiogenic phenotype, an effect that can be reduced with nSMase2 inhibition [64]. Tumor EVs often carry proteins and miRNAs that encourage tumor growth and metastasis, such as polyadenylate-binding protein 1 and miR-21-5; nSMase2 inhibition dramatically reduces their release and, thus, their protumor influence [65,66]. Tumor-derived EVs have also been shown to increase proliferation and limit apoptosis in both melanoma and colon cancer cells, an effect that was reduced following GW4869 treatment [67,68]. In scratch assays, migration of colorectal cancer (CRC) cells was attenuated by nSMase2 inhibition [69], while nSMase2 inhibition enhanced the inhibitory effects of metapristone on the migration of cervical cancer cells [70]. Media from cancer-associated fibroblasts (CAFs) were found capable of inducing an epithelial-to-mesenchymal transition (EMT), a hallmark of metastasis, in several human lung cancer cell lines; the nSMase inhibitor GW4869 blocked EV release from fibroblasts and prevented the isolated media from inducing EMT in recipient cells [71]. Not only are tumor-derived EVs affected by nSMase2 inhibition, but EVs derived from tumor-associated macrophages, which can drive metastasis, are also reduced, with GW4869 treatment leading to reduced metastasis [72]. Small cell lung cancer (SCLC) typically metastasizes to the brain and it was determined that EVs released by brain microvascular endothelial cells promoted the expression of S100 A16, a marker associated with brain metastases, in SCLC cells [73]. Inhibiting EV release from brain microvascular cells prevented increased S100 A16 expression and reduced survival in SCLC cells in response to H₂O₂ [73]. EVs isolated from Ca²⁺-dependent activator protein for secretion 1 (CAPS1)-overexpressing CRC cells enhanced migration of normal fetal colonic mucosal cells, but GW4869 significantly blocked this migration [74].

Drug resistance

In addition to influencing tumorigenesis and metastases, tumor-derived EVs have also been implicated in the induction of drug resistance [63]. Tumor-derived EVs are capable of transferring drug-resistance mechanisms to sensitive cells in a handful of cancer cell types, and nSMase2 inhibition can abrogate this process. For example, CAFs have been shown to release EVs that promote gemcitabine resistance in pancreatic ductal adenocarcinoma cells; GW4869 was shown to block the EV release and limit the resistance [75]. An adverse effect of paclitaxel and other chemotherapeutic agents is inducing senescence, allowing tumor cells to evade apoptosis and thereby creating a reservoir of cells that can provide an environment encouraging metastasis or can reactivate and metastasize themselves. Paclitaxel-induced senescent breast cancer cells have enhanced EV release, but nSMase2 inhibition blocks this enhanced released and reduces senescence-associated β-galactosidase (SA-β-Gal) expression and cell survival [76]. In another study, cisplatin-resistant oral squamous cell carcinoma cells were shown to release EVs containing miRNA-21, which induces cisplatin resistance in naïve recipient cells. When GW4869 was used to block the release of EVs from donor cells, EV-recipient cells exhibited reduced resistance [77]. Drug resistance to bortezomib in a murine model of multiple myeloma was also alleviated with GW4869 [78]. In human CRC cells, inhibition of miRNA-19b-containing EVs with nSMase2 inhibition led to decreased oxaliplatin resistance and enhanced cell death [79]. EVs secreted by gastric cancer cells and carrying miR-105-5p were found to elicit doxorubicin resistance, whereas treating the parent cells with GW4869 limited the transfer of resistance [80]. Several prostate cancer cells lines that are resistant to the androgen receptor inhibitor enzalutamide were found to have increased EV release, and inhibiting this release with GW4869 enhanced cell death [81]. Although most publications implicate enhanced EV release and nSMase2 in drug resistance, some report the opposite. For example, in human breast cancer cells, enhancing the release of EVs containing the breast cancer resistant protein (BCRP) was found to reduce doxorubicin resistance [82], highlighting the importance of evaluating the role of EVs and nSMase2 inhibition in drug resistance in individual cancers and against specific chemotherapies.

Immunotherapy

Another important role for tumor-derived EVs is modulating the tumor immune response. One of the mechanisms by which tumors evade the immune system is by upregulating Programmed death-ligand 1 (PD-L1), a cell surface immune checkpoint ligand that binds to PD-1 on effector T cells and dampens their response. PD-L1 has been observed on tumor-derived EVs from several different cancers and is able to interact with effector T cells and dampen their antitumor response [83]. Co-treatment of GW4869 with anti-PD-L1 antibody therapy improved the antitumor efficacy versus anti-PD-L1 antibody alone in a breast cancer model [84]. In a recent study by Poggio et al., the removal of EV PD-L1 via CRISPR/Cas9 inhibited tumor growth in the TRAMP-C2 murine model of prostate cancer [85]. Furthermore, injection of PD-L1-null or nSMase2-null TRAMP-C2 cells failed to generate tumors in mice, whereas injection of PD-L1-null TRAMP-C2 cells promoted T cell activation [85]. Remarkably, when WT TRAMP-C2 cells were injected in one flank of a mouse simultaneously with PD-L1-null or nSMase2-null TRAMP-C2 cells on the other flank, the wild-type tumors failed to grow, supporting the idea that immune cells activated on the PD-L1 or nSMase2-null side were able to migrate and attack the wild-type tumor cells on the opposite flank. These data suggest nSMase2 inhibition as an effective therapeutic combination with immunotherapy to enhance systemic antitumor immunity.

Conclusion

The role of nSMase2-dependent EVs in cancer is complex and varies between cancers, cargo content, and chemotherapies. Whereas some studies demonstrated a beneficial role nSMase2 in limiting cancer progression, the majority of literature points to nSMase2-mediated EV biogenesis as a mediator of tumorigenesis, metastasis, and drug resistance, with a role in diminishing antitumor immunity response, making it a logical therapeutic target in several cancers.

Utility of nSMase2 inhibition in inflammation

Over the past decade, the role of EVs in exacerbating inflammatory responses to injury and disease has gained attention, and the effectiveness of nSMase2-mediated inhibition of EV biogenesis and release on these conditions is just beginning to be explored (Table 4).

Table 4.

Utility of nSMase2 inhibition in preclinical models of inflammatory diseases

Disease	Model	Result of nSMase2 inhibition	Refs
Airway inflammation	Intratracheal LPS	Improved lung elastance, decreased inflammation	[88]
Allergic airway inflammation	BEAS-2B cells; ovalbumin-sensitized mice	Decreased EV secretion in lung cells and lung inflammation	[86]
	House dust mite-sensitized mice	Decreased airway-secreted EVs, Th2 cytokines, and eosinophils	[87]
Atherosclerosis	Mesenteric small artery extracts	Decreased markers of vascular inflammation (VCAM)	[94]
	DC-HUVEC co-culture	Decreased EV release and activation of HUVECs	[95]
	APOE−/− mice	Decreased atherosclerotic lesions, macrophage infiltration, and lipid deposition	[96]
	LDLR−/− mice	Reduced atherosclerosis, glucose, lipids, insulin resistance, and hepatic steatosis	[97]
Cerebral ischemia	Focal cerebral ischemia	Inhibited EV release and decreased neuroinflammation	[90]
	Transient global ischemia	Decreased hippocampal ceramide levels, neuronal damage, and inflammation	[91]
Hepatic IR injury	Rat hepatic IR injury	Decreased liver COX activity and PGE2 levels	[89]
Myocardial infarction	Myocardial infarction	Reduced tachypnea, diaphragm dysfunction, and ceramide levels	[92]
Sepsis	Cecal ligation puncture	Decreased calpain activation and diaphragm weakness	[98]
	Endotoxin or cecal ligation puncture	Inhibited EV release, decreased inflammation, and prolonged survival	[93]

Inflammatory airway diseases

EVs are involved in the inflammatory response to airway diseases. For example, EVs isolated from bronchial epithelial cells have been shown to induce monocyte chemotaxis and proliferation [86]. In a mouse model of allergic airway inflammation, treatment with the nSMase2 inhibitor GW4869 led to fewer lung macrophages and improved airway hyper-responsiveness and bronchial pathology [86]. In house dust mite-sensitized mice, nSMase2 inhibition reduced the number of EVs in bronchoalveolar lavage fluid, resulting in fewer eosinophils in the lungs as well as declines in interleukin (IL)-4 and IL-13 [87]. In an LPS-induced mouse model of airway inflammation and injury, blockade of nSMase2 also reduced proinflammatory cytokines, IL-6 and IL-1β, and improved lung elastance, alveolar collapse, and immune cell infiltration, but whether these findings were the result of reduced EV release was not explored [88].

Ischemia-reperfusion injury and sepsis

Whereas aSMase has historically been the focus in ischemia-reperfusion (IR) injury, several publications report that inhibiting nSMase2 can also improve outcomes. In a liver IR model, GW4869 improved histopathological scores and decreased cyclooxygenase and prostaglandin E2 [89]. In preclinical cerebral ischemia models, blocking proinflammatory EV release from brain tissue with GW4869 resulted in fewer Iba1+ cells in the cortex and hippocampus and a shift in microglia from the proinflammatory state to anti-inflammatory state, as measured by a decrease in CD86 and increase in CD206 levels [90] and a reduction in inflammatory markers [91]. nSMase2 inhibition was also shown to reduce tachypnea and diaphragm dysfunction in a myocardial infarction model [92]. nSMase2-mediated EV release has also been implicated in the ‘cytokine storm’ phenomenon seen in sepsis. EVs isolated from LPS-stimulated macrophages were shown to contain enhanced levels of proinflammatory cytokines, which were capable of inducing further cytokine release from naïve macrophages [93]. In preclinical LPS-induced inflammation and cecal ligation puncture sepsis models, nSMase2 inhibition reduced myocardial inflammation and proinflammatory cytokine levels, improved cardiac function, and prolonged survival

Atherosclerosis

Chronic endothelial inflammation is implicated is atherosclerosis. In patients with hypertension, endothelin-1 is elevated and activates nSMase2, which increases vascular cell adhesion protein 1 (VCAM-1) and vascular inflammation, leading to small artery remodeling [94]. Inhibiting nSMase2 with GW4869 lowers VCAM-1 expression in rat mesenteric small arteries [94]. In addition to nSMase2 itself playing a part in vascular inflammation, EVs released from dendritic cells (DCs) contain tumor necrosis factor (TNF)-α and are capable of inducing activation of cultured human umbilical vein endothelial cells (HUVECs) via nuclear factor (NF)-κB signaling [95]. When DC EVs were inhibited by nSMase2 inhibition, the conditioned media could no longer activate endothelial cells, thereby significantly reducing levels of VCAM-1, intercellular cell adhesion protein 1 (ICAM-1), and E-selectin [95]. In addition to reducing pathogenic EVs, nSMase2 inhibition attenuated inflammatory responses and beneficially curtailed endothelial cell activation in vitro. This approach also decreased atherosclerotic lesion area in APOE^−/− mice, an effect observed similarly following genetic depletion of nSMase2 or treatment with GW4869 [96]. Atherosclerotic plaque formation was also attenuated with nSMase2 inhibition in mice fed a high-fat diet before LPS treatment [97]. Interestingly, this same study also observed atherosclerotic plaque decreases with aSMase inhibition [97].

Conclusion

Taken together, there is mounting evidence that nSMase2 inhibition has a role in modulating multiple aspects of the inflammatory response and its inhibition might have therapeutic applications for myriad inflammatory diseases.

nSMase2 in infectious diseases

Inhibition of nSMase2 is being explored as a therapeutic target for bacterial and viral infections. Both nSMase2 and EVs in general have been implicated in multiple infectious diseases, including HIV, Zika virus, rabies virus, Dengue virus (DENV), and Shiga toxin (Table 5).

Table 5.

Utility of nSMase2 inhibition in preclinical infectious disease models

Disease	Model	Result of nSMase2 inhibition	Refs
CMV	Primary human dermal fibroblasts	Decreased propagation of HCMV	[108]
DENV	C6/36 cells	Decreased EVs and viral load	[106]
Epsilon toxin	ACHN cells	Inhibited toxin-mediated ceramide formation and cytotoxicity	[110]
HCV	Huh7-Lunet-TLR3 cells	Decreased secretion of infectious virus	[103]
HEV	PLC/PRF/5 cells	Decreased secretion of infectious virus	[104]
HIV	DCs and CD4+ T cells	Blocked trans-infection from HIV-1-infected cells to healthy DCs and CD4+ T lymphocytes	[100]
Langat virus	ISE6 and HaCaT cells	Inhibited release of tick cell-derived EVs and decreased viral RNA transmission	[105]
NDV	DF-1 cells	Decreased EV release from infected cells and decreased extracellular levels of virus	[109]
Rabies virus	Vero cells	Decreased levels of viral RNA	[107]
Shiga toxin	THP-1 cells	Decreased toxin-mediated human renal cell death	[111]
Zika virus	Human fetal astrocytes	Decreased EV release and suppressed virus propagation	[101]
	Primary murine cortical neurons	Reduced EV release and diminished viral RNA levels	[102]

Viral infections

EVs are implicated in the propagation of HIV infection (reviewed in [99]). EVs released from HIV-infected cells carry HIV accessory proteins and co-receptors that make target cells more receptive to HIV infection. Additionally, the virion can physically associate with EVs, which can enable it to evade immune surveillance and increase infectivity. EVs from HIV-1-infected CD4+ T cells can also induce HIV-1 reactivation from dormant viral reservoirs in resting CD4+ T lymphocytes [100]. In cell culture experiments, blocking the release of EVs from infected CD4+ T cells with the nSMase2 inhibitors GW4869 and spiroepoxide reduced DC-mediated infection of healthy CD4+ T lymphocytes [100].

In addition to HIV, nSMase2 inhibitors have also shown therapeutic promise against the Zika virus. Zika infection in human fetal astrocytes increased the release of EVs and viral particles; some of the viral particles were packaged within the EVs. Inhibiting EV release via GW4869 led to diminished Zika virus propagation [101]. Similar findings were observed in murine neuronal cell cultures, where Zika virus led to enhanced EV release containing viral RNA. Either silencing nSMase2 using siRNA or pharmacologically inhibiting the enzyme with GW4869 reduced EV release and diminished viral RNA levels [102].

The efficacy of nSMase2 inhibitors has also been explored in Hepatitis C (HCV), Hepatitis E (HEV), Rabies, Langat virus (LGTV), DENV, cytomegalovirus (HCMV), and Newcastle disease virus (NDV). In HCV, double-stranded RNA was found in EVs released from infected hepatocytes; blocking EV release using GW4869 resulted in reduced viral replication [103]. Similarly, HEV particles were packaged into EVs and released from infected liver cells. Again, the EV release of HEV was decreased following nSMase2 inhibition [104]. A tick-borne flavivirus, LGTV, which is similar to tick-borne encephalitis virus, was found within EVs from both arthropod and mammalian cells. Inhibiting EV release lessened EV viral RNA loading and viral plaque formation [105]. Infectious RNA of another flavivirus, the mosquito-borne DENV, was also found in EVs released from DENV-infected mosquito-derived cell lines, and treatment with GW4869 partially blocked transmission to human cells [106]. Similarly, extracellular viral RNA, likely associated with EVs from rabies virus-infected cells, was also reduced following GW4869 treatment [107]. The human herpes virus, HCMV, was also found to utilize EVs for propagation, which was slowed by nSMase2 inhibition [108]. Interestingly, viral proteins packaged into EVs are not unique to mammalian infectious diseases because proteins of NDV, a poultry virus, were detected in EVs released from infected chicken fibroblasts, and GW4869 treatment lowered infectivity [109].

Bacterial infections

The effects of nSMase2 inhibition on bacterial infections are less studied than for viral infections, but a few publications have demonstrated utility. Epsilon-toxin produced by Clostridium perfringens, a lethal bacterial infection of undulates, was shown to enhance ceramide production in exposed kidney cells. Treatment of the exposed kidney cells with GW4869 reduced cell death [110]. The bacterial Shiga toxin, released by certain strains of Escherichia coli associated with GI, kidney, and central nervous system (CNS) pathology, was found to be packaged into EVs derived from exposed macrophages. These EVs induced cell death in naïve HK-2 renal epithelial cells. Renal epithelial cell death rates were ameliorated when EV release was blocked with nSMase2 inhibition [111].

Conclusion

There are strong indications that EVs are involved in the propagation and pathogenesis of viral infections, while preliminary evidence suggests their involvement in bacterial toxin-induced pathology. These areas represent exciting and possibly wide-reaching therapeutic applications for nSMase2 inhibitors.

Concluding remarks and future directions

This review summarizes the rapidly developing field of ceramide-dependent EVs as they relate to disease, with an emphasis on therapeutic opportunities afforded by nSMase2 inhibition. Although there is expert consensus that EVs contribute to the pathogenesis of a variety of diseases, caution must be exerted in unraveling the diverse biological networks that these EVs influence. First, EV isolation techniques are not standardized, and each technique demonstrates a bias toward certain subpopulations of EVs and varying degrees of non-EV contaminants [4]. As such, it is important to keep in mind the isolation technique used and any biases that might confound the results of the study when interpreting results. Second, several reports point to a beneficial role for ceramide-dependent EVs in diseases such as ALS [55], retinal degeneration [112], inflammatory bowel disease [113], and AD [114], which are in direct opposition to the mostly pathological role of these EVs reviewed earlier. It is probable that cell and cargo type, along with the stage of disease, dictates whether EVs are helpful or harmful, further complicating the application of nSMase2 inhibitors as therapeutic modalities. Adding to this, EVs isolated from umbilical cord mesenchymal stem cells have beneficial anti-inflammatory effects, highlighting both their role in maintaining homeostasis and their potential to be utilized as immunomodulatory therapeutics [115]. As EV separation and characterization techniques improve [116], we will be able to better assess their involvement in health and disease.

EVs also have a central role in neurodevelopmental processes [117] and nSMase2-KO mice have disrupted hypothalamic–pituitary–adrenal axes, leading to dwarfism, defects in bone formation, and overall hypoplasia [25]. These data warn that administration of nSMase2 inhibitors during childhood could be detrimental. In normal mice, one report showed that inhibition of nSMase2 worsened cognitive function [118], suggesting that disruption of EV function when there is no disease burden causes toxicity. Overall, there is much work to be done before nSMase2 inhibitors are available in the clinic, but existing research suggests that initial utility should be limited to mature adults diagnosed with diseases in which there is clear evidence that EVs contribute to pathological burden.

Although numerous nSMase2 inhibitors and genetic knockdown studies have demonstrated benefit in preclinical animal models of disease, the major impediment to clinical translation is the lack of a potent, drug-like inhibitor with good pharmacokinetic and safety profiles. Better compounds will also provide the ability to further understand the often complicated roles of EVs in disease. Given several exciting recent advances, such as the discovery of PDDC and DPTIP [37,38,40,41], there is optimism that a clinic-ready nSMase2 inhibitor is near, and we are buoyed with the knowledge that a growing number of research teams are engaged in exploring the therapeutic applications of this fascinating enzyme.

Research highlights:

• Extracellular vesicles (EVs) are potent vehicles of intercellular communication

• EVs can transport pathological cargo that contributes to disease

• One pathway of EV biogenesis is dependent upon ceramides generated by nSMase2

• Inhibition of nSMase2 shows promise in treating diseases that propagate via EVs

• Potent and selective nSMase2 inhibitors have recently been discovered

Biography

Carolyn Tallon completed her BSc in physiology at McGill University before earning her PhD in pathobiology at Johns Hopkins University. She completed her doctoral thesis work in the lab of Mohamed Farah determining the efficacy of BACE1 inhibitors on enhancing peripheral nerve regeneration in injury and disease. She is currently a postdoctoral fellow in the lab of Barbara Slusher at Johns Hopkins University studying the efficacy of small-molecule inhibitors in neurodegenerative diseases, particularly neutral sphingomyelinase 2 inhibitors in Alzheimer’s disease.

Barbara Slusher is a professor of neurology, pharmacology, psychiatry, neuroscience, medicine and oncology at Johns Hopkins School of Medicine and the Director of Johns Hopkins Drug Discovery. Before joining Johns Hopkins, Dr Slusher spent 18 years in the pharmaceutical industry, and has extensive experience in drug discovery through early clinical development. In 2009, she joined Hopkins to lead a small-molecule drug discovery program with a veteran team of over 25 medicinal chemists, assay developers, pharmacologists/toxicologists, and pharmacokinetics/drug metabolism experts. The team is engaged in identifying novel drug targets and translating them into new drug therapies for clinical development.